Development of an LC–MS–MS method for the quantification of taurine derivatives in marine invertebrates

doi:10.1016/j.ab.2004.06.014

Analytical Biochemistry

Volume 332, Issue 2, 15 September 2004, Pages 215-225

https://doi.org/10.1016/j.ab.2004.06.014 Get rights and content

Abstract

Sulfur amino acids, such as taurine, hypotaurine, and thiotaurine, were found in high quantities in tissues of marine symbiotic organisms (e.g., bivalves, tubeworms) living close to hydrothermal vent sites. Therefore, they are assumed to play a key role in the S-oxidizing base metabolism or sulfide detoxification. We propose here a specific, rapid, and original analytical procedure for the direct determination of sulfur amino acids at the level of a few parts per billion in biological samples, avoiding the classical low specific post-column ortho-phthaldialdehyde derivatization step required by non-ultraviolet-absorbing molecules. Indeed, by coupling liquid chromatography on a porous graphitic stationary phase under isocratic conditions (10 mM ammonium acetate buffer adjusted to pH 9.3) to tandem mass spectrometry (ionization process by pneumatically assisted electrospray in negative ion mode), it is possible to perform specific quantification of these metabolites in less than 10 min directly in biological matrices without any derivatization step or other tedious sample treatments. Thus, taurine, hypotaurine, and thiotaurine have been identified and assayed in several deep sea organisms, showing that the developed method is well suited for this kind of application.

Section snippets

Reagents

Taurine (NH₂–CH₂–CH₂–SO₃H) and hypotaurine (NH₂–CH₂–CH₂–SO₂H) were purchased from Sigma, 2-aminoethyl hydrogenosulfuric acid (NH₂–CH₂–CH₂–O–SO₃H) was purchased from Fluka, and 2-aminoethylthiosulfonic acid (NH₂–CH₂–CH₂–S–SO₃H) was purchased from Aldrich. Pure crystalline thiotaurine (NH₂–CH₂–CH₂–SO₂SH) was prepared according to the method of Cavallini et al. [32], whereby a solution of hypotaurine in 0.2 N NaOH is reacted with elemental sulfur in ethanol for 2 h at 85 °C. The solution is then

Results and discussion

Taurine-like compounds (Table 1) can be considered as amino acids. Thus, LC–MS–MS was investigated to replace the lengthy traditional LC–UV or fluorescence methods using pre- or postderivatization (mainly ortho-phthaldialdehyde) of the analytes. The first step in the development of the LC–MS or LC–MS–MS analytical methodology was the investigation of the most specific and sensitive conditions for detection (e.g., ionization mode, fragmentation).

Conclusion

The goal of this study was to develop a reliable analytical method to characterize and quantify unusual sulfur amino compounds, especially thiotaurine, found in symbiotic deep marine invertebrates. Coupling LC with tandem MS proves to be a reliable procedure for the identification and quantification of metabolites in biological samples given that MS–MS detection is a highly sensitive and specific detection mode, whereas LC provides selectivity between the remaining isobars or isomers contained

Acknowledgments

We thank the scientific teams and the crew of the joint French–U.S. hydrothermal vent cruise Hydronaut for their assistance in collecting material. This study was partially supported by a financial Grant (02/CNES/4800000093) from the Centre National d’Etudes Spatiales (CNES).

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Hypotaurine, a precursor to taurine, is known for its antioxidant properties and is prominently present in fetal plasma and the placenta. Our previous research revealed that ezrin-knockout mice experience fetal growth retardation, coinciding with reduced hypotaurine levels in fetal plasma. This study aims to elucidate the expression and role of hypotaurine transporters within the placenta.
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Among hypotaurine transporters, GAT2 exhibited increased mRNA and protein expression in murine placenta during mid-to-late gestation. Notably, GAT2/Slc6a13 mRNA and hypotaurine were most concentrated in the labyrinth of murine placenta. In contrast, enzymes responsible for hypotaurine synthesis, such as cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and 2-aminoethanethiol dioxygenase, showed minimal expression in the labyrinth. These findings suggest that GAT2 is a key determinant of hypotaurine levels in the placental labyrinth. Immunohistochemical examination unveiled that GAT2 was predominantly localized on the fetal-facing plasma membrane within syncytiotrophoblasts, which co-localized with ezrin. In rat umbilical perfusion experiments, the GAT2/3 and TauT inhibitor, SNAP-5114, significantly reduced hypotaurine extraction from fetal circulation to the placenta.
The results suggest that GAT2 plays a pivotal role in the concentrative uptake of hypotaurine from fetal plasma within syncytiotrophoblasts of the placenta.
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High sensitivity (0.5 nM for taurine) was achieved from microchip electrophoretic separation using a combination of field-amplified stacking and reversed-field stacking [9]. There are also reported methods without the derivatization step that apply conductivity [2], amperometric [4] or mass spectrometry (MS) detection techniques [6–8]. The capillary electrophoresis (CE) method with contactless conductivity detection for the determination of taurine in energy drinks [2] is rapid method and very sensitive.
An electrophoretic method (on-line coupled capillary isotachophoresis and capillary zone electrophoresis) with conductometric detection for the determination of free taurine in selected food and feed is described. Taurine is converted to isethionic acid by van Slyke method. Under optimized conditions (leading electrolyte: 5 mM HCl, 10 mM glycylglycine, and 0.05% 2-hydroxyethyl cellulose solution, pH 3.2; terminating electrolyte: 10 mM citric acid; background electrolyte: 50 mM acetic acid, 20 mM glycylglycine, and 0.1% 2-hydroxyethyl cellulose solution, pH 3.7), isethionic acid is separated from other sample components in anionic mode and detected using a conductimeter within 15 minutes. The performance method characteristics, such as linearity (25 – 1250 ng/mL), accuracy (99 ± 9%), repeatability (3.9%), reproducibility (4.3%), limits of detection (3 ng/mL) and quantification (10 ng/mL) were evaluated. By analysing 20 food and pet food samples the method was proved suitable for routine analysis. High sensitivity and selectivity, short analysis time and low costs are significant features of the presented method.
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The first four metabolites showing significant changes in serum were found to be ascorbic acid, tryptophan, tyrosine, and phenylalanine. In our study, targeted metabolomic analyses were performed on human plasma samples by using LC-ESI-MS/MS. For this purpose, a suitable LC-ESI-MS/MS method was developed and validated that allowed for the analysis of twenty six biomarkers (glutamic acid [30], lactic acid [31], histidine [32], N-acetyl glycine [16], fumaric acid [33], azelaic acid [16], 2-aminoisobutyric acid [34], choline [35], glutamine [30], taurine [36], proline [37], 3-hydroxybutyric acid [38], diethylaminomalonic acid, glucose [39], glyceric acid [40], malic acid [41], tyrosine [42], 5-hydroxymethyl-2-deoxyuridine [43], citrulline [44], glucuronic acid [45], uracil [46], phosphocholine [47], o-phosphoethanolamine [48], xanthine [49], 2-aminoadipic acid [50], and serine [51]) selected for the early diagnosis of breast cancer. The developed method was employed to analyze the plasma samples of both control and patient groups, leading to the identification of new biomarker candidates for the early detection of breast cancer.
The best preventive method for breast cancer, which accounts for 18% of the most common cancer-related deaths among women, is early diagnosis. Metabolomic analysis plays an important role in both early diagnosis and tracking of the disease progress. In this study, targeted metabolomic analyses were performed on twenty-six selected metabolites, which were proposed in the literature for use in the early diagnosis of breast cancer, by examining the plasma samples of 105 healthy volunteers, 172 early stage and 92 metastatic breast cancer patients. To this purpose, an LC-ESI-MS/MS method was developed, validated, and applied to the analysis of the above plasma samples. Measurements were performed on a Merck SeQuant ZIC-HILIC column (50 × 4.6 mm, 5 µm) with the mobile phase run in the gradient mode with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL min⁻¹ and 40 °C. The correlation coefficient values between 0.991 and 0.999 in the study range where each metabolite is linear shows the linearity of the method. The LOD and LLOQ values of the metabolites were between 5.9 × 10⁻⁵-1.0 × 10⁻² μg mL⁻¹ and 1.8 × 10⁻⁴-5.3 × 10⁻² μg mL⁻¹, respectively. The validation studies revealed that the method was linear, sensitive, precise, accurate, and selective. Our results showed that the metabolite identification based exclusively on the m/z values obtained from untargeted metabolomic studies must conform with standards before their clinical usage. Since only nine (citrulline, malic acid, glyceric acid, 3-hydroxybutyric acid, glutamine, proline, choline, fumaric acid and lactic acid) out of the twenty-six metabolites changed significantly among breast cancer patients. Therefore untargeted metabolomic analyses should be confirmed by targeted analyses before suggesting metabolites as biomarkers to avoid possible mismatches.
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Previous study reported that ABC transporters took part in absorption, distribution and elimination processes (Scherrmann, 2009). Besides, taurine could also serve as the key mediator of sulphur metabolism and functioned in the intracellular isosmotic regulation in marine invertebrates (Chaimbault et al., 2004). Accordingly, we deduced that ABC transporters may be involved in the stabilization of intracellular environment in mud crab.
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Sulfur containing amino acids and related compounds was quantified from algal extracts with a Shimadzu UFLC-XR (Shiamadzu Scientific Instruments, Inc., Columbia, MD) and an AB SCIEX Q-Trap 4000 LC–MS/MS (AB SCIEX, Framingham, MA) electron spray ionization detector. Isocratic chromatographic separations were achieved within 8 min using a Zorbax Eclipse plus 3.5 μm, 150 × 4.6 mm reverse phase C18 column (Agilent Technologies, Santa Clara, CA) and a 10 mM aqueous ammonium acetate solution (pH 9.3) [20] at a constant flow rate of 0.3 mL min− 1 and 40 °C column temperature. The column was washed for 2 min with methanol between sample runs and allowed to re-equilibrate for 3 min prior to the next injection to remove photosynthetic pigments.
Taurine (2-aminoethanesulfonic acid) is an amino acid-like compound widely distributed in animals and an essential nutrient in some species. Targeted metabolomics of marine and fresh water microalgae combined with medium supplementation identified biosynthetic pathway intermediates and necessary catalytic activities. Genomic analysis was then used to predict the first taurine biosynthetic pathway in these organisms. MRM-based electrospray ionization (ESI) LC–MS/MS analysis demonstrated that taurine is synthesized using a carbon backbone from l-serine combined with sulfur derived from sulfate. Metabolite analysis showed a non-uniform pattern in levels of pathway intermediates that were both species and supplement-dependent. While increased culture salinity raised taurine levels modestly in marine alga, taurine levels were strongly induced in a fresh water species implicating taurine as an organic osmolyte. Conservation of the synthetic pathway in algae and metazoans together with a pattern of intermittent distribution in other lineages suggests that it arose early in eukaryotic evolution. Elevated levels of cell-associated taurine in algae could offer a new and biorenewable source of this unusual bioactive compound.
Simultaneous determination of taurine, glucuronolactone and glucuronic acid in energy drinks by ultra high performance liquid chromatography-tandem mass spectrometry (triple quadrupole)
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On the contrary, there are many analytical methods available in literature for taurine quantification, i.e. by using Fourier transform infrared spectroscopy (FTIR) [11] or nuclear magnetic resonance (NMR) [12] techniques. Due to absence of strong absorbing groups, taurine is quantified by means of HPLC with pre-column derivatization and fluorescent detector [13–15], or with pre-column derivatization and HPLC/MS [16,17] or with HPTLC with post-chromatographic derivatization [18]. To our best knowledge, no chromatographic analytical methods are available in literature for taurine quantification without derivatization steps.
In this work, we present for the first time a rapid and robust UHPLC–MS/MS method for analyzing taurine, GlcLA and GlcA in energy drinks simultaneously and without derivatization. The separation of three analytes was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d.) and a mobile phase formed by water (A) and acetonitrile (B) both with formic acid 0.1% at a flow rate of 0.8 ml min⁻¹ with isocratic elution in 3.5 min. Calibration curves were calculated using the method of standard addition in a concentration range from 2 to 6 mg/100 ml for taurine (R² > 0.987), from 0.4 to 1.2 mg/100 ml for GlcLa (R² > 0.997), and from 0.2 to 0.6 mg/100 ml for GlcA acid (R² > 0.998). The validated method was applied to the analysis of nine commercial energy drinks. The level of taurine found ranged from 0.01 to 0.45 g/100 ml, and it matched with that reported in the labels of the analyzed energy drink samples.

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Development of an LC–MS–MS method for the quantification of taurine derivatives in marine invertebrates

Abstract

Section snippets

Reagents

Results and discussion

Conclusion

Acknowledgments

Prog. Oceanogr.

Comp. Biochem. Physiol. A

J. Chromatogr.

J. Chromatogr. A

J. Chromatogr. B

J. Chromatogr. B

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Neurochem. Intl.

Geomicrobiology of deep-sea hydrothermal vents

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